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New Phytol.195, 844–56.
Seasonal trends in the biomass and structure of the bryophyte-associated fungal communities explored by 454 pyrosequencing2012

Davey, M.L., Heegaard, E., Halvorsen, R., Ohlson, M. and Kauserud, H.

Notes: Genomic DNA was extracted from shoot fragments using an organic extraction procedure and purified using the Wizard® SV Gel and PCR Clean-Up System prior to template preparation by nested PCR. Products from the second PCR were separated by electrophoresis and purified using the Wizard® SV Gel and PCR Clean-Up System prior to pyrosequencing. (4548)

Notes: These authors investigated the role of matrix metalloproteinase (MMP-9) in a reversible model of cancer that is initiated by infection with intracellular Theileria parasites. They found that gene induction by parasite infection was associated with trimethylation of histone H3K4 (H3K4me3) at the MMP-9 promoter. The H3K4 methyltransferase SMYD3 was the only histone methyltransferase upregulated upon infection. They therefore investigated the role of SMYD3 overexpression on MMP-9 expression and cell migration, identifying SMYD3 as an important new regulator of MMP-9 transcription. During the study they used the GloMax® Multi Luminometer to measure luminescence and absorbance in reporter and cell viability assays. They also used the Dual-Luciferase® Reporter Assay to measure SMYD3 activity in cells transfected with a SMYD3 reporter, and the pGL4-hRluc/TK plasmid for normalization of the experimental reporter activity. GoTaq® DNA polymerase was used in semi-quantitative RT-PCR. (4189)

Notes: In this report, the original investigators who found XMRV and pMLV (polytropic murine leukemia virus) in blood of subjects with Chronic Fatigue Syndrome (CFS) report that this association is not confirmed in a blinded analysis of samples from rigorously characterized subjects.

The CDC performed nucleic acid testing assays. Plasma was centrifuged and RNA isolated from the pellet. Quantitative real-time RT-PCR assays (qRT-PCR) for generic pMLV/XMRV pro (protease) and gag detection were performed on RNA extracts, using the AccessQuick™ RT-PCR System and an AgPath one-step RT-PCR kit.

ArrayScript RT and AmpliTaq Gold DNA polymerase were used for cDNA synthesis and amplification in the pro and gag qRT-PCR assays, respectively. A third PCR was done using the primers XPOLOF and XPOLOR, followed by a nested PCR with the primers XPOLIF and XPOLIR for the generic detection of MLV/XMRV 216-bp pol sequences. For this reaction, cDNA synthesis and amplification of RNA was done using Promega AMV Reverse Transcriptase and a RobustI RT-PCR kit. Each PCR experiment included 20 water-only reactions to control for contamination. (4300)

Notes: These authors used the Roche 454 next generation sequencing platform to sequence and compare cancerous and normal horn tissue transcripts. mRNA isolated from each sample was fragmented prior to cDNA synthesis. cDNA quality was verified using a high sensitivity DNA Chip kit on the Bioanalyzer 2100 and the QuantiFluor™-ST Fluorometer. The transcripts were compared and potential tumor associated genes identified. (4229)

Notes: Polyglutamine diseases are neurodegenerative disorders associated with the presence of proteins containing polyglutamine repeats. These authors studied the mechanism of polygluatmine toxicity. Mutant RNAs carrying an expanded CAG repeat were shown to activate the nucleolar stress pathway and induce apoptosis. Expanded CAG RNAs were shown to interact with nucleolin, preventing it from binding to an upstream control element of the rRNA promoter and causing decreased rRNA transcription, which in turn induced apoptosis. Perturbations in rRNA transcription were identified by real-time PCR, and fluorescence in situ hybridization was used to determine that expanded CAG RNAs localized to the nucleolus. Hybridization solutions were supplemented with RNasin® Ribonuclease Inhibitor. ImProm-II™ Reverse Transcriptase was used in RT-qPCR assays. (4228)

Notes: This study examined the prevalence of strains of human rhinovirus (HRV) that may be causing respiratory infections in Mexican children. Nucleic acids were purified from nasal swabs of two-year-old children, and screened for the presence of HRV by amplifying 20ng of HRV-RNA using the AccessQuick™ RT-PCR System with primers for the 5´ nontranslated region. Products were sequenced and aligned with sequences found in GenBank. (4343)

Food Control23, 400-404.
Development and validation of fast Real-Time PCR assays for species identification in raw and cooked meat mixtures.2012

Cammà, C., Di Domenico, M., and Monaco, F.

Notes: These authors used the Maxwell&reg; 16 Tissue DNA Purification Kit to extract DNA from 200µl samples of raw meat homogenate from beef, pork, poultry and lamb samples. They also used the Wizard® Genomic DNA Isolation System to extract DNA from cooked meat samples. The extracted DNA was used in real-time, quantitative PCR assays to identify species-specific DNA spiked at 1% in mixed DNA samples. (4353)

Notes: Oropouche fever is a arboviral infection in Brazil, surpassed in frequency only by dengue. Oropouche virus (OROV) causes large outbreaks of acute febrile illness in areas along the Amazon and Central-Plateau regions. RNA was extracted from CSF and underwent reverse transcription-polymerase chain reaction and sequencing to identify OROV. Reverse transciption was performed with 5ml of the random primers, using the AccessQuick™ RT-PCR System. (4320)

Notes: This study examined how well primers developed in 1990 and 2004 for type A rotavirus (RVA) were able to genotype (G type) currently circulating RVAs in Asia. The VP7 gene from RVA was amplified using 2µl of dsRNA template with the AccessQuick™ RT-PCR System in a total volume of 50µl. The G type was determined using hemi-nested multiplex PCR using 1µl of the VP7 cDNA and PCR Master Mix in a final volume of 50µl. The final products were sequenced. (4340)

Notes: This study describes the clinical observations and laboratory investigations performed on 170 of the 2,000 suspected West Niles Virus (WNV) cases. These cases were admitted to Aravind Eye Hospital, Madurai, Tamil Nadu with ocular complications. Conventional reverse transcription PCR (RT-PCR) and real-time RT-PCR assays were used to detect WNV infection. In addition, reverse transcription loop-mediated isothermal gene amplification (RT-LAMP) was performed to determine the feasibility of using this method as an alternative cost-effective tool to the real-time RT-PCR.

After proving negative for DENV- and CHIKV, samples were tested for the presence of WNV-specific RNA by RT-PCR, real-time RT-PCR and RT-LAMP assays. RNA was extracted from the patient serum, plasma and infected culture supernatant. The RNA was then eluted in 50µl of nuclease-free water and used as template in the AccessQuick™ RT-PCR System, with primer pairs targeting the env gene.

Amplification was performed in a 50µl total reaction volume with the AccessQuick™ RT-PCR System, using 50pmol of each forward and reverse primer and 2µl of extracted viral RNA. (4331)

Notes: Mice RNA samples were converted to cDNA using an oligo-dT primer with the Reverse Transcription System, ethanol precipitated, vacuum dried and transferred to another lab. There they were reconstituted in 20μl of molecular biology grade water.

Detection of caliciviruses in the wild mice samples was attempted using generic calicivirus primers targeting sequences encoding conserved amino acid motifs in the RNA-dependent RNA polymerase (RdRp) region of ORF1. Two microliters of cDNA was used in 25μl PCR reactions using the GoTaq® Green Master Mix. Laboratory mouse RNA samples were tested only with MNV-specific primers in the AccessQuick™ RT-PCR System using 2μl RNA as template.

PCR products were cloned into pGEM-T® Vector and sequenced using M13 forward and reverse primers on an ABI PRISM® 3730 DNA Analyzer. (4330)

Notes: The authors previously showed that there are two independent mechanisms by which Chlamydia-specific CD4 T cells clear infection in epithelial cells; an iNOS-dependent mechanism and a Plac8-dependent mechanism. To further identify the Plac8 mechanism, they used microarrays to identify a second mechanism dependent on Plac8 for terminating Chlamydia replication in epithelial cells.

Several Chlamydia-specific CD4 T cell clones were purified at the end of their culture cycle and grown for 3 days in their usual culture media plus growth factors, without Ag stimulation. Total RNA was isolated from each clone using a protocol that included an RNase-free DNase I treatment step. Specific mRNA gene reverse transcription and amplification were performed using the AccessQuick™ RT-PCR System. (4324)

Notes: This paper describes a method for sequencing unknown viral isolates from tissue culture using anchored random reverse transcription and PCR, pyrosequencing and data analysis. RNA was extracted from tissue culture supernatants positive for viral antigens and used in RT-PCR with random primers. Amplification products were gel-purified and used in pyrosequencing reactions. A QuantiFluor™-P Fluorometer was used to measure copy number concentration relative to a standard, prior to Roche 454 pyrosequencing. (4231)

Appl. Environ. Microbiol.78, 445–54.
Responses of methanogen mcrA genes and their transcripts to an alternate dry/wet cycle of paddy field soil.2012

Ma, K., Conrad, R. and Lu, Y.

Notes: The authors of this study investigated the microbial mechanisms associated with the reduction of methane production and emission from rice fields observed with intermittent field drainage. They looked in particular at the abundance of mcrA gene copies and transcripts from rice paddy soil fauna. The mcrA gene encodes the alpha subunit of methyl coenzyme M reductase.

Total nucleic acid was extracted from soil samples using a phenol-chloroform procedure. For RNA analyses, DNA was hydrolyzed using RQ1 RNase-free DNase in the presence of 0.2µl Recombinant RNasin® Ribonuclease Inhibitor and then further purified using a commercial kit. cDNA synthesis was carried out using the Improm-II™ Reverse Transcription System, again in the presence of 1.0µl Recombinant RNasin® Ribonuclease Inhibitor. A clone library of transcripts was generated using the pGEM®-T Easy Vector System. The transcript standard for quantitative mcrA analysis was prepared from the in vitro transcript of a mcrA clone using the Riboprobe® in vitro Transcription Systems. (4241)

Notes: In this study, the authors evaluated whether application of the phytochemical shikonin to the skin of mice was able to augment the effect of a DNA-based anti-tumor vaccine by inducing the cytokine RANTES. As part of the study, the AccessQuick™ System was used in RT-PCR analysis to determine expression of RANTES mRNA in treated and control skin samples. (4288)

Notes: Swab samples from 11 species of Chinese bats were vortexed in maintenance medium, filtered through a 0.45µm pore filter, centrifuged and resuspended. Any nonencapsidated (naked) nucleic acid was digested in a cocktail of enzymes including 20U of RNase ONE™ Ribonuclease prior to DNA and RNA purification. Conserved regions of the RNA-dependent RNA polymerase gene of astroviruses were reverse transcribed, amplified and cloned into the pGEM®-T Easy Vector for sequencing. (4552)

Notes: The authors used GoTaq® DNA Polymerase to amplify cDNA generated from total RNA (RT-PCR) extracted from murine erythroid leukemia (MEL) cells and mouse erythroid burst-forming units (BFU-Es). These cells were used to study the molecular mechanism of 5-aza-2'-deoxycytidine (5-aza-CdR)-induced erythroid differentiation, a process involved in azanucleotides for treating myelodysplastic syndromes (MDS) that reduces the risk of transformation to acute myeloid leukemia (AML). Treatment of these cells with 5-aza-CdR, a hypomethylation reagent, upregulated genes responsible for heme production and iron uptake. The pGL3 basic vector and promoter were used to create plasmid constructs of different E-box regulatory sequences with a luciferase reporter. The plasmids were cotransfected with c-Myc, Max or both transcription factors into human hepatocytes (HepG2). The Dual-Luciferase® Reporter Assay System was used to identify that the –6kb E-box of the transferrin receptor 1 (TfR1) promoter was a strong enhancer for inducing TfR1 expression when c-Myc and Max formed functional complexes that bound to it. Bisulfite sequencing was performed to study methylation patterns after 5-aza-2’-CdR treatment using the pGEM-T® Easy Vector system to ligate the isolated DNA fragments for TfR1 and Fech (ferrochetalase), which were transformed into E coli. for final sequencing. (4176)

Notes: DNA was isolated from a variety of environmental samples including surface soil, drinking water biofilm, sludge from an anerobic digester, bioreactor samples, ground water, peat soil and glacial deposit soil. The 16S rRNA gene was amplified from the DNA. PCR amplifications were run on agarose gels, and bands of the predicted sizes excised and purified using the Wizard® SV Gel and PCR Clean-Up System before pooling for pyrosequencing. (4554)

Notes: Sputum samples were collected from cystic fibrosis patients and 16S rRNA sequences amplified by PCR. These products were cloned into a T-vector, transformed into competent cells and the resulting colonies grown in 2ml LB broth in 96-deep-well plate for 20 hours. Of this culture, 1.9ml was pelleted and the clones isolated using the Wizard® SV Plasmid Purification System. The purified plasmid DNA was subjected to agarose gel electrophoresis and sequenced. (4133)

Notes: The authors of this study sought to correlate promoter methylation with gene expression. Gene expression data was generated by RNA-seq of Jurkat cells. Amplified cDNA was prepared from total RNA, the cDNA was treated with S1 nuclease to remove single stranded nucleic acids and used as template for the sequencing libraries. (4536)

Notes: This proof-of-principle study investigated whether attenuated Salmonella typhimurium could be used as a vehicle for delivering shRNA-expressing plasmid DNA into cancer cells in mice. The authors delivered S. typhimurium bearing plasmids encoding anti-GFP shRNA orally to mice harboring tumors that espressed GFP. They were able to show that the bacteria accumulated and persisted for 40 days within the tumors, and that GFP expression in infected tumors was reduced. The AccessQuick™ RT-PCR System was used to analyze GFP expression levels in cultured cells and tumors. (4347)

Notes: These authors showed that expression of the serine protease inhibitor elafin is regulated by epigenetically controlled expression of the transcription factor Foxa2. Treatment of melanoma cells with a DNA methyltransferase inhibitor induced elafin expression, resulting in reduced proliferation and increased apoptosis. Luciferase reporter assays were used to show that Foxa2 binding was required for activation of elafin expression, and that Foxa2 binding was activated by treatment with the methyltransferase inhibitor. These assays used a pGL3-Basic Vector construct in which expression of firefly luciferase was driven by the upstream region bearing the Foxa2 binding site. The pRL-TK Vector, expressing Renilla luciferase, was used as a normalization control. The AccessQuick™ System was used for RT-PCR analysis to show that Foxa2 mRNA was barely detectable in melanoma cells. (4345)

Cytokine55, 79-89.
GM-CSF plays a key role in zymosan-stimulated human dendritic cells for activation of Th1 and Th17 cells.2011

Wei, W.C., Su, Y.H., Chen, S.S., Sheu, J.H., and Yang, N.S.

Notes: This study compared the effects of zymosan and LPS on human monocyte-derived dendritic cells (DCs). The authors found that zymosan-activated DCs had a unigue cytokine expression profile. In zymosan-activated DCs, high levels of GM-CSF and IL-27, rather than IL-12 p70, were involved in Th1 cell activation. As part of the study, RT-PCR was used to investigate the molecular basis of this failure to induce production of active IL-12 p70. Expression levels of the biologically inactive subunits of IL-12 p70 (p40 and p35) was assessed using the AccessQuick™ RT-PCR System. The results showed that zymosan induced expression of p40, but not p35 mRNA, indicating that lack of induction of p35 was the reason for failure to induce active IL-12 p70. (4348)

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